The general class of small analytes which are to be assayed includes small molecular weight (about 10,000 daltons or less, preferably about 6,000 daltons or less, which is less than the size usable in a sandwich assay format, and which is capable of being recognized and bound by a ligand) pesticides, drugs, toxins, mycotoxins and drug metabolites, and further includes trichothecenes, fumonisins, antibiotics, and fragments of microorganisms, such as bacteria, parasites, viruses, yeast and mold, and fragments of mammalian cells. In particular, non-invasive screening procedures for assessing the exposure of humans to these substances require the ability to quantify both the target analyte and in some cases, metabolites, such as hydroxylated derivatives which include Aflatoxin M1, derived by hydroxylation from Aflatoxin B1, and/or other metabolites in body fluids such as milk, serum and urine.
Both the parent and the present inventions relate to a small analyte detecting assay which is novel because, in one test, it incorporates the best aspects of Enzyme Linked Immunosorbent Assay (ELISA) and Immunoaffinity chromatography (IA), but is not one or the other, since in contrast to ELISA, neither the parent nor the present invention use an enzyme signaling method, and in contrast to IA, the parent and present inventions depend on a competition and, therefore, do not result in isolation solely of purified target analyte, nor in isolation of target analyte at high efficiency. In addition, the inventions can be applied as general signal generation methods for a variety of target analytes.
Previously, with each new target analyte, a visualization approach specific to that target analyte was needed, which requires great expenditure of Research and Development resources. In the majority of cases this problem is solved by the parent invention which is drawn to a generic signaling assay method that is readily adaptable to most target analytes. However, some small analytes are hydrophobic and hard to chemically modify or difficult to work with within the context of the parent invention. The present invention overcomes the problems associated with this sub-set of small analytes while conserving the approach of the parent invention, resulting in greater efficiency of non-invasive immunological screening procedures and reduced Research and Development costs.
Finally, the method of the present invention is highly sensitive, i.e. has a very low detection limit, and yet is more rugged than other methods (such as enzyme-based methods) because the method of the invention has a longer shelf-life and is more forgiving in the hands of a user.
The invention uses an affinity matrix material for detecting a small analyte target comprising a solid phase sorbent material and a first ligand such as a monoclonal or polyclonal antibody, or a non-antibody ligand specific for the target analyte as well as a conjugated version of the analyte, in which the first ligand is bound to the sorbent material. This aspect of the invention provides a novel and widely useful method of testing for the presence of a small analyte by use of a ligand that binds to both the small analyte and conjugated small analyte, in which the ligand is bound to a solid phase sorbent material. Examples of solid phase sorbent materials are; sepharose and other agarose gel compositions, dextrans, magnetic beads or particles, charged nylon membranes, carbon and silicon granular preparations and the like, including glass beads and plates.
The invention can be summarized as follows. Any small target analyte and any conjugate decorated with multiple target analytes which are capable of competitively binding a first antibody or specific ligand that is linked to a solid support matrix can be employed. A second antibody or specific ligand which recognizes both the analyte and the multiple-analyte-decorated conjugate, and that has a fluorescent label, or any chemical compound that serves as a direct label, including colorimetric and isotopic labels, but specifically excluding all enzymes and indirect signal generators, is used. A sample that is spiked with the multiple-analyte-decorated conjugate is exposed simultaneously, or sequentially, in either order, to the first ligand and the second, tagged ligand. The amount of the label detected indicates the concentration of the endogenous amount of the analyte in the sample in inverse relationship as described below.
A test sample is mixed or may be incubated with a pre-determined amount of the multiple-analyte-decorated conjugate. When the test sample containing the multiple-analyte-decorated conjugate is passed over the immunoaffinity matrix, the first antibodies can bind competitively to two species: free analyte and multiple-analyte-decorated conjugate. The matrix is then exposed to a second ligand, such as a mono- or polyclonal antibody that is tagged or fluorescently labeled. Either within the matrix or upon elution, high label presence is seen in a clean sample. Conversely, only a small amount of the tag or label is detected in the matrix or eluant of a test sample that is highly contaminated with the free analyte.
It is an object of this invention to provide a kit for rapidly and accurately determining the presence or absence of small analytes in a sample quantitatively or non-quantitatively as desired. Each component of the kit(s) may be individually packaged in its own suitable container. The individual containers may also be labeled in a manner which identifies the contents. Moreover, the individually packaged components may be placed in a larger container capable of holding all desired components. Associated with the kit may be instructions that explain how to use the kit. These instructions may be written on or attached to the kit.
The present invention is drawn to a small analyte that is less than 10,000 Da, which is tagged with a non-enzyme label.
The present invention is further drawn to an affinity matrix for the detection of a small analyte comprising:
a solid phase sorbent material; and
a first ligand which is specific for both said small analyte and the small analyte which is conjugated or tagged with a non-enzyme label, wherein said first ligand is immobilized on said sorbent material.
The present invention is further drawn to a method for detecting small analytes in a test sample which comprises the steps of:
exposing a sample believed to contain a small analyte in combination with a predetermined amount of said analyte that decorates a non-enzyme conjugate, to a solid phase sorbent material that has immobilized thereon a first ligand that is specific for both said analyte and said multiple-analyte-decorated conjugate;
washing said solid phase sorbent material to remove non-specifically associated sample material;
exposing said solid phase sorbent material to a second ligand which is specific for said analyte and said multiple-analyte-decorated conjugate, and which is tagged or labeled;
washing said solid phase sorbent material to remove non-specifically associated tagged second ligand;
detecting the presence and amount of said tagged second ligand, for example, by subjecting said resin to UV light to induce fluorescence.
In addition, the present invention is further drawn to a method for detecting small analytes in a test sample which comprises the steps of:
exposing a sample believed to contain a small analyte in combination with a predetermined amount of said analyte that decorates a non-enzyme conjugate, to a solid phase sorbent material that has immobilized thereon a first ligand that is specific for both said analyte and said multiple-analyte-decorated conjugate;
washing said solid phase sorbent material to remove non-specifically associated sample material;
exposing said solid phase sorbent material to a second ligand which is specific for said analyte and said analyte-decorated conjugate, and which is tagged or labeled;
washing said solid phase sorbent material to remove non-specifically associated tagged second ligand;
exposing said solid phase sorbent material to a releasing agent, whereby said analyte, analyte-decorated conjugate and tagged second ligand are released from said first ligand;
recovering said analyte, multiple-analyte-decorated conjugate and tagged second ligand in an eluant; and
detecting the presence and amount of said tagged second ligand in said eluant, for example, by subjecting said eluant to UV light to induce fluorescence.
In addition, the present invention is further drawn to a kit for detecting small analytes in a test sample, comprising:
a solid phase sorbent conjugated to a first ligand specific for both a small analyte of molecular weight of not more than about 10,000 daltons and said analyte that decorates a conjugate;
a multiple-analyte-decorated conjugate;
a second tagged ligand to be detected that is specific for both said small analyte and said multiple-analyte-decorated conjugate;
instructions for carrying out the detection method.
The present invention is based on the observation that free small analytes as described below, when bound to an antibody or ligand are incapable of binding a second antibody or ligand, unlike larger molecules. In this embodiment of the present invention, a target small analyte and a known quantity of said small analyte decorating a conjugate, compete for binding of an immobilized first antibody or ligand specific to said small analyte and said multiple-analyte-decorated conjugate. After removing unbound material, the immobilized first antibody or ligand with bound small analytes and multiple-analyte-decorated conjugates is exposed to a second, tagged antibody or ligand which is specific to said small analyte and said multiple-analyte-decorated conjugate. Multiple-analyte-decorated conjugates are able to bind the second, tagged antibody or ligand while free small analytes are too small and are, thus, unable to participate in these second binding reactions. The amount of label then detected indicates the concentration of the endogenous amount of the analyte in the sample in an inverse relationship.
The present invention is useful for the detection and isolation of a small analyte generally when it presents two characteristics or, for antibody based assays, three characteristics: first, the target analyte has a molecular weight not greater than about 10,000 daltons; second, in situations where the antibody ligand is used, that the target analyte, either alone or in combination with other compounds, is usually able to induce an immunological response in vivo when introduced into an animal subject; and third that the ligand is capable of recognizing both the free target analyte and a tagged or conjugated analyte.
The first characteristic of a molecular weight not greater than about 10,000 daltons, is easily determinable for any target analyte, including a toxic substance.
The second characteristic, in situations where the antibody ligand is used, the ability of the small analyte to induce an antibody response after introduction into a test animal in vivo, relies on its ability primarily (but not exclusively) to serve as an immunogen.
The third characteristic, the capability of the ligand to recognize the free target analyte as well as the multiple-analyte-decorated conjugate, is easily determinable for any substance and is within the skill of the person of ordinary skill in the art.
The analyte is less than about 10,000 Da, preferably about 6,000 Da, more preferably about 3,000 Da, and most preferably 1,000 Da or less. The analyte is particularly small enough so as to not function in a sandwich assay, i.e. the analyte is too small to be simultaneously bound by two different ligands. The decorated conjugate is comprised of a substance which itself has at least two target anayltes attached thereto, the substance being a protein (such as bovine serum albumin (BSA), ovalbumin (OA), keyhole limpet chemocyanin (KLH) or an antibody), but also including carbohydrates (such as poly- or oligo-saccharides (including glucose or other sugar polymers, pectin, amylose, malto-dextrin or limit-dextrin)), DNA and peptido-nucleic acid (PNA). For example, T-2 toxin is a molecule of less than 10,000 Da. Two or more T-2 toxin molecules can be bound to one BSA molecule to create a xe2x80x9cdecorated conjugatexe2x80x9d. In a second form, a xe2x80x9cdecorated conjugatexe2x80x9d could be two T-2 toxin molecules attached to a protein or sugar oligomer serving as a linker and being large enough so that two independent binding sites for T-2 toxin are created, but not so large as to create another binding site independent of the T-2 toxin molecules. The term xe2x80x9cligandxe2x80x9d as used herein includes any molecule that specifically recognizes and binds both the free analyte and multiple-analyte-decorated conjugate. A xe2x80x9cligandxe2x80x9d can refer to components of a polyclonal anti-serum, monoclonal antibody, or a non-antibody molecule.
The term xe2x80x9cnon-antibody ligandxe2x80x9d as used herein includes non-antibody molecules that can be used to bind the target analyte as well as the multiple-analyte-decorated conjugate of the present invention. Such ligands include, for example, lectins and receptors such as opiate receptors.
The term xe2x80x9clabelxe2x80x9d or xe2x80x9ctagxe2x80x9d as used herein includes all non-enzymatic compounds that serve as a reporter of the presence of the ligand to which it is bound, and does not prevent the binding of the analyte or the multiple-analyte-decorated conjugate to the tagged ligand. Examples of such labels include those which are radioactive, fluorescent, chemiluminescent, colored or absorbent, or a combination of the foregoing.
Radioactive or isotopic labels include, for example, 14C 3H, 35S, 125I and 32p. Fluorescent labels include any compound that emits an electromagnetic radiation, preferably visible light, resulting from the absorption of incident radiation and persisting as long as the stimulating radiation is continued. Such compounds include coumarin containing molecules, and further include anthroyl compounds, naphthalene compounds, pyrene compounds, compounds containing benzyl, pyrenyl and phenyl groups, fluorescein compounds, anthracene compounds, compounds containing conjugated pi electron systems, but are not limited to these categories of compounds and include any compound that could be used as a label in this invention.